The technique of radioimmunoassay had its inception approximately twenty years ago in the study of plasma insulin in human subjects by immunological methods.
Conventional radioimmunoassays are characterized by the necessity for a series of incubation periods whose duration is dependent upon the type of assay being conducted and the type of antigen being subjected to the assay. The necessity for as many as three incubation periods naturally prolongs the time necessitated for the completion of the study. Thus, the final results of the assay are frequently not available until the day following the initiation of the procedure, since some radioimmunoassays take as long as twenty-four hours to complete.
The time lapse occasioned by conventional radioimmunoassays is particularly critical in such cases as cardiac arrest where the subject is placed in intensive care and there may be doubt as to the exact nature of the patient's problem until the results of the radioimmunoassay are made available.
It is not unknown for a patient to succumb before the results of the assay are available which, if available at an earilier time, might have conceivably sharpened the diagnosis and permitted the utilization of techniques which would have saved the life of the patient.
It is well known that many seeming heart arrests are really caused by acute indigestion or vice versa. Frequently, the exact nature of the disorder cannot be determined until the results of the time-consuming radioimmunoassay have been received.
The reason for the lengthy incubation periods lies in the fact that there is an ambivalent hunt and seek of the labelled antigen and unlabelled or serum antigens for sites on the antibody to which the antigens are exposed. Therefore, it takes considerable time for the process to reach comparative stabilization in which the results can be obtained by exposure of the incubated mixture to a gamma count.
For instance, in a standard immunoassay, a standard reference or unknown antigen is admixed in aqueous suspension with a specific antibody and subjected to a first period of incubation. The resultant suspension is then admixed with 125.sub.I labelled antigen and a second incubation occurs. The resultant suspension is then admixed with a second antibody or charcoal or ammonium sulfate and is subjected to a third incubation. The completion of the third incubation results in the precipitation of antibody with bound components which are separated by configuration or filtration and the isolated precipitate is then subjected to radioactivity measurement by the use of gamma counters or similar instruments.
A second common procedure involves only the first two incubations of the previously discussed procedure and instead of utilizing a second antibody or equivalent substances, protein A/Staphylococcus aureus is admixed with the product of the second incubation. The steps of the method than include precipitation, separation and measure of radioactivity of isolated precipitate.
In a third form of radioimmunoassay the first incubation occurs with the specific antibody chemically bound to the surface of a particle, test tube or the like. First and second incubations occur as in the previously discussed methods but the resultant suspension of the second incubation is subjected to the binding of a proportion of antigen on the surface-bound antibody. Then the separation and radioactivity measurement steps take place.
As can be readily determined, incubation steps are an inherent part of all of the known methods and the deleterious delay resulting from such necessary incubation steps can result in misdiagnosis and possible consequent loss of life of the patient. Corrollary factors to the necessity for prolonged incubation are the expense of the radioimmunoassay, the amount of wastage of various ingredients including the isotope labelled antigens and the possible error in the assay resulting from the lack of complete binding of antigens on the antibody.
Typical of prior art developments in U.S. Pat. No. 4,107,284 issued Aug. 18, 1978 on a radioimmunoassay procedure using a stabilized complex. The above-referenced patent constituted an attempt to extend the shelf life of a complex of labelled antigen and antibody by the utilization of stabilizers.
Basically, the conventional methodology can be divided into two procedures. First is the mixing of standard, reference or unknown antigens with a specific antibody. Second is the mixing of the first mix with the 125.sub.I labelled antigen.
In the first mixture, unlabelled antigens are added to specific antibody. The labelled antigen (tracer) is then added and competes with the unknown, nonlabelled antigen for the binding sites on the antibody. The amount of unknown antigen is thereby inversely proportional to the amount of labelled antigen which is bound to the antibody. Usually, simultaneous procedures are conducted for standard antigens (unlabelled antigens of known concentration) in order to derive a standard curve. The concentration of unknown antigen can then be determined by reference to the standard curve.
When the labelled antigen is added to the antigen-antibody complex, a gradual equilibrium occurs in which the unknown or standard antigen competes and exchanges with the labelled antigen for the binding sites on the antibody. The procedure normally requires that the unknown antigen and the standard antigen behave identically in this ability to compete with and/or displace labelled antigen from specific antibody, but identical behavior between the labelled antigen and the unknown and standard antigen are not required. In conclusion, at least one and usually two lengthy incubations or reagents are required in the first procedure.
Following incubation to form an antigen-antibody complex, the free and bound components are separated. The usual procedure for separation employs a second antibody which has a specific affinity for the first antibody and forms a precipitate. The precipitate is isolated from the free components by centrifugation and decanting the solution containing the free components.
Other methods have been devised to facilitate the separation of antigen-antibody complex from unbound antigen. Many of these involve the binding of antibody to a solid phase, e.g. Sephadex particles, glass beads, plastic beads, test tube surfaces, filter paper, etc.
Another, more recent method for separation of free and bound components employs protein A associated with the cell wall of the bacterium Staphylococcus aureus. Protein A has a high affinity for the Fc fragments of IgG molecules of several mammalian species. Thus, these bacteria can be used in place of the second antibody to form a complex with the antigen-antibody complex which can then be isolated by centrifugation and decanting. The reaction rate between the bacterial protein A and IgG is very rapid in comparison to the double antibody technique and represents a substantial advantage.
However, two significant problems are associated with the use of the protein A bearing Staphylococcus aureus in this alternative method. First, large amounts of protein A are required since the protein A must remove all of the IgG from the serum sample as well as the IgG of the first antibody. This results in a significant cost penalty. Second, the large amount of bacteria needed for the assay reslts in a very high nonspecific binding of labelled antigen which significantly increases the error of the assay.